Multiple amplification cycle detection

ABSTRACT

Methods and devices are provided for simultaneously amplifying a plurality of sample wells for a predetermined amount of amplification, detecting whether amplification has occurred in a first set of the wells, amplifying for an additional amount of amplification and detecting whether amplification has occurred in a second set of the wells. Methods are also provided for analyzing a target nucleic acid sequence using melt curves that were generated in a plurality of amplification cycles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/037,948, filed Jul. 17, 2018, which is a divisional of U.S.application Ser. No. 14/425,588, filed Mar. 3, 2015, which issued asU.S. Pat. No. 10,053,726 on Aug. 21, 2018, which is a 35 USC § 371national phase application of International Application Serial No.PCT/US2013/058752, filed Sep. 9, 2013, which claims the benefit of andpriority to U.S. Provisional Application No. 61/699,103, filed on Sep.10, 2012, entitled “Multiple Amplification Cycle Detection,” the entirecontents of each of which are incorporated herein by reference.

GOVERNMENT INTEREST

This invention was made with government support under Grant No. 1U01AI082184 awarded by National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays to be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. A challengeto using PCR as a primary means of diagnosis is the variety of possiblecausative organisms and the low levels of organism present in somepathological specimens. It is often impractical to run large panels ofPCR assays, one for each possible causative organism, most of which areexpected to be negative. The problem is exacerbated when pathogennucleic acid is at low concentration and requires a large volume ofsample to gather adequate reaction templates. In some cases, there isinadequate sample to assay for all possible etiological agents. Asolution is to run “multiplex PCR” wherein the sample is concurrentlyassayed for multiple targets in a single reaction. While multiplex PCRhas proven to be valuable in some systems, shortcomings exist concerningrobustness of high level multiplex reactions and difficulties for clearanalysis of multiple products. To solve these problems, the assay may besubsequently divided into multiple secondary PCRs. Nesting secondaryreactions within the primary product often increases robustness.However, this further handling can be expensive and may lead tocontamination or other problems.

The FilmArray® (BioFire Diagnostics, Inc., Salt Lake City, Utah) is auser friendly, highly multiplexed PCR system developed for thediagnostic market. The single sample instrument accepts a diagnostic“pouch” that integrates sample preparation and nested multiplex PCR.Integrated sample preparation provides ease-of-use, while the highlymultiplexed PCR provides both the sensitivity of PCR and the ability totest for up to 30 different organisms simultaneously. This system iswell suited to pathogen identification where a number of differentpathogens all manifest similar clinical symptoms. Current availablediagnostic panels include a respiratory panel for upper respiratoryinfections and a blood culture panel for blood stream infections. Otherpanels are in development.

Many of the organisms that are targeted in FilmArray panels, as well asin panels for use with other instruments, are commonly present in theenvironment. While such environmental contamination tends to be presentin concentrations that are significantly below that of a clinicallyrelevant sample, it can be difficult to distinguish betweenenvironmental contamination and clinical infection. Also, certainindividuals have latent viral infections through chromosomalintegration, wherein the chromosomally integrated viral DNA may or maynot be responsible for the clinical symptoms. It would be desirable tohave methods for determining whether a positive result is due to aclinically relevant infection or due to another source of nucleic acid.

SUMMARY OF THE INVENTION

The present disclosure relates to methods for simultaneously amplifyinga number of targets, while distinguishing between clinically relevantamplification and amplification from other sources such as frombackground contamination, cross-reactivity in the amplificationreaction, or chromosomal integration.

In one aspect of the present invention methods and devices foridentifying which of a plurality of target nucleic acids is in a sampleare disclosed. The disclosed methods comprise providing a plurality ofsample wells, each sample well provided with primers for amplifying alocus from a different one of the plurality of target nucleic acidsequence, providing a portion of the sample into each of the pluralityof sample wells, simultaneously subjecting the plurality of sample wellsto amplification conditions through a number of amplification cycles,detecting whether amplification has occurred in each of a first set ofthe plurality of sample wells, simultaneously subjecting the pluralityof sample wells to amplification conditions through a number ofadditional amplification cycles, detecting whether amplification hasoccurred in each of a second set of the plurality of sample wells, andidentifying the target nucleic acid present in the sample by identifyingthe corresponding sample well in which amplification has occurred.

In another illustrative embodiment, methods are provided fordistinguishing between chromosomal integration and clinically-relevantinfection in a sample, illustratively comprising providing a sample wellprovided with the sample and primers for amplifying a target nucleicacid sequence from the sample, subjecting the sample well toamplification conditions through a number of amplification cycles,detecting whether amplification has occurred in the sample well,subjecting the sample well to amplification conditions through a numberof additional amplification cycles, and detecting whether amplificationhas occurred in the sample well. In certain illustrative examples, apositive call in the first detecting step may be indicative ofchromosomal integration, and a negative call in the first detecting stepwith a positive call in the second detecting step may be indicative of aclinically-relevant infection.

In yet another illustrative embodiment, methods for analyzing a targetnucleic acid in a sample are provided comprising

(a) providing a sample well comprising therein the sample and primersfor amplifying the target nucleic acid,

(b) subjecting the sample well to amplification conditions through a atleast one amplification cycle,

(c) generating a melt curve of the amplified target nucleic acid, and

(d) repeating steps (b) and (c).

These methods may further include determining a value for the meltcurve, and determining a Cp by identifying the amplification cycle inwhich the value for the melt curve exceeds a predetermined value. Inillustrative examples, the value may be determined by peak height orpeak area of a negative derivative of the melt curve.

In still another example, methods for analyzing a target nucleic acid ina sample are provided comprising

(a) providing a sample well including the sample, primers for amplifyingthe target nucleic acid, a control nucleic acid, primers for amplifyingthe control nucleic acid, and a dsDNA binding dye,

(b) subjecting the sample well to amplification conditions through aplurality of amplification cycles,

(c) generating a melt curve of the amplified target nucleic acid andamplified control nucleic acid,

(d) determining a value for the amplified target nucleic acid, and

(e) repeating steps (b), (c), and (d).

Such illustrative methods may also include the step of generating acorrected amplification curve for the target nucleic acid using thevalues determined in step (d), and plotting corrected amplificationcurves, or determining relative concentrations between the two nucleicacids.

Reaction vessels and devices are also provided herein. Additionalfeatures of the present invention will become apparent to those skilledin the art upon consideration of the following detailed description ofpreferred embodiments exemplifying the best mode of carrying out theinvention as presently perceived.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an illustrative pouch for use in one embodiment in thisdisclosure.

FIGS. 2A-B show amplification and melt curves after three differentcycles for A. baumannii. FIG. 2A shows data for a false positive andFIG. 2B shows data for a true positive.

FIGS. 3A-B show amplification and melt curves after three differentcycles for C. tropicalis. FIG. 3A shows data for a negative sample andFIG. 3B shows data for a positive sample.

FIGS. 4A-B show amplification and melt curves after three differentcycles for S. aureus. FIG. 4A shows data for a negative sample and FIG.4B shows data for a positive sample.

FIG. 5 shows an illustrative cycling protocol for detecting low loadsamples.

FIG. 6 shows illustrative amplification curves and cut-off fluorescentthreshold.

FIG. 7 shows illustrative temperature data that may be collected duringa typical two-step PCR protocol. During the denaturation/annealingsegment, the temperature is increased from the baseline value to themaximum, followed by a decrease in temperature back to the baseline.During the extension segment, the temperature is held constant.

FIG. 8 shows illustrative continuous monitoring of fluorescence datathat may be collected during the two-step PCR protocol of FIG. 7.

FIG. 9 shows illustrative continuous monitoring of fluorescence datathat may be collected during two cycles of a typical two-step PCRprotocol. During the denaturation segment, the fluorescence datadecreases as the saturating dsDNA-binding dye is released from thedsDNA, resembling a typical melt curve. During the extension segment,the fluorescence data increases as the primed ssDNA fragments are primedand extended into dsDNA fragments.

FIG. 10 shows an overlay of illustrative fluorescence data that may becollected during the denaturation segments for several cycles of PCR.

FIG. 11 shows an overlay of illustrative the negative first derivativeof the fluorescence data collected during the denaturation segments forseveral cycles of PCR.

FIG. 12 shows a typical amplification curve for a multiplexed PCRreaction that includes a control nucleic acid and a target nucleic acidof unknown concentration.

FIG. 13 shows an overlay of illustrative negative first derivative offluorescence data that may be collected during the denaturation segmentsfor several cycles of PCR of a multiplex reaction containing a controlnucleic acid and a target nucleic acid.

FIG. 14 shows illustrative adjusted real-time PCR curves for the controland sample nucleic acids of FIG. 13. By integrating the negative firstderivative of the melt curves generated by continuous monitoring the PCRreaction over the control window, an adjusted amplification curve forthe control nucleic acid is generated (solid line). By integrating thenegative first derivative of the melt curves generated by continuousmonitoring the PCR reaction over the target window, an adjustedamplification curve for the target nucleic acid is generated (dashedline).

FIG. 15 illustrates a block diagram of an exemplary embodiment of athermal cycling system in accordance with aspects of the disclosure.

DETAILED DESCRIPTION

As used herein, the terms “a,” “an,” and “the” are defined to mean oneor more and include the plural unless the context is inappropriate.Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 5%. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; a solution containing one or moremolecules derived from a cell, cellular material, or viral material(e.g. a polypeptide or nucleic acid); or a solution containing anon-naturally occurring nucleic acid, which is assayed as describedherein. A sample may also be any body fluid or excretion (for example,but not limited to, blood, urine, stool, saliva, tears, bile,cerebrospinal fluid) that contains cells, cell components, or nucleicacids.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combinationthereof

By “probe,” “primer,” or “oligonucleotide” is meant a single-strandedDNA or RNA molecule of defined sequence that can base-pair to a secondDNA or RNA molecule that contains a complementary sequence (the“target”). The stability of the resulting hybrid depends upon thelength, GC content, and the extent of the base-pairing that occurs. Theextent of base-pairing is affected by parameters such as the degree ofcomplementarily between the probe and target molecules and the degree ofstringency of the hybridization conditions. The degree of hybridizationstringency is affected by parameters such as temperature, saltconcentration, and the concentration of organic molecules such asformamide, and is determined by methods known to one skilled in the art.Probes, primers, and oligonucleotides may be detectably-labeled, eitherradioactively, fluorescently, or non-radioactively, by methodswell-known to those skilled in the art. dsDNA binding dyes may be usedto detect dsDNA. It is understood that a “primer” is specificallyconfigured to be extended by a polymerase, whereas a “probe” or“oligonucleotide” may or may not be so configured.

By “dsDNA binding dyes” is meant dyes that fluoresce differentially whenbound to double-stranded DNA than when bound to single-stranded DNA orfree in solution, usually by fluorescing more strongly. While referenceis made to dsDNA binding dyes, it is understood that any suitable dyemay be used herein, with some non-limiting illustrative dyes describedin U.S. Pat. No. 7,387,887, herein incorporated by reference. Othersignal producing substances may be used for detecting nucleic acidamplification and melting, illustratively enzymes, antibodies, etc., asare known in the art.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a sample nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant conditions that allowhybridization comparable with that resulting from the use of a DNA probeof at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well known by thoseskilled in the art of molecular biology.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependent amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. For amplification methodswithout discrete cycles, reaction time may be used where measurementsare made in cycles or Cp, and additional reaction time may be addedwhere additional PCR cycles are added in the embodiments describedherein. It is understood that protocols may need to be adjustedaccordingly.

Emerging technologies, such as multiplex PCR or MALDI-TOF, are capableof rapidly detecting numerous bacterial and viral pathogens that impacthuman health. By simultaneously screening for multiple pathogens, thesetechnologies save time and money by reducing the number of laboratorytests required for diagnosis. One challenge with rapid, broad-spectrumyet specific detection is that not all pathogens are present atidentical titers. For example, in positive blood cultures Gram-negativebacteria typically grow to higher titers while yeast grows more slowlyand to lower titers. This is further complicated by the potential ofdetecting background environmental organisms. Air, soil, dust, andhumans are all carriers of bacterial organisms. Moreover, the test kititself may contain trace nucleic acids, even if the test kit and itscontents have been sterilized. Also, where organisms are cultured, thegrowth media often contains non-viable organisms, which would not affectculture, but could produce false positives in PCR. If a system isdesigned uniformly for increased sensitivity to detect low titerspathogens, frequent false positive results may occur from backgroundorganisms. Alternatively, if system sensitivity is reduced to avoidbackground organism detection, low titer organisms may be missed,resulting in false negative detection. Two-step multiplex PCR protocolsenable detection over a broad range of titers, but this broad range ofdetection can make it difficult to distinguish between a true positiveand a minor environmental contaminant. By individually tuning the numberof PCR cycles performed before detection of each assay, low titertargets can be readily detected in the same multiplex PCR reaction ashigher target organisms, while minimizing false positive calls frombackground contamination, cross-reactivity (which can be problematic ina highly multiplexed reaction), and other extraneous amplification.

Various embodiments disclosed herein use a self-contained nucleic acidanalysis pouch to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. Such systems, includingpouches and instruments for use with the pouches, are disclosed in moredetail in U.S. Pat. No. 8,394,608; U.S. Patent Application No.2010-0056383; and WO 2013/074391, herein incorporated by reference.However, it is understood that such pouches are illustrative only, andthe multiple PCR reactions discussed herein may be performed in any of avariety of open or closed system sample vessels as are known in the art,including 96-well plates, plates of other configurations, arrays,carousels, and the like, using a variety of amplification systems, asare known in the art. While the term “sample well” is used herein, thisterm is meant to encompass wells, tubes, and various other reactioncontainers, as are used in these amplification systems. In oneembodiment, the pouch is used to assay for multiple pathogens.Illustratively, various steps may be performed in the optionallydisposable pouch, including nucleic acid preparation, primary largevolume multiplex PCR, dilution of primary amplification product, andsecondary PCR, culminating with optional real-time detection orpost-amplification analysis such as melting-curve analysis. Further, itis understood that while the various steps may be performed in pouchesof the present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.

FIG. 1 shows an illustrative pouch 510 for use with the currentinvention. Pouch 510 is similar to FIG. 15 of U.S. Patent ApplicationNo. 2010-0056383, already incorporated by reference, with like itemsnumbered the same. Fitment 590 is provided with entry channels 515 athrough 515 l, which also serve as reagent reservoirs. Illustratively,reagents may be freeze dried in fitment 590 and rehydrated prior to use.Blisters 522, 544, 546, 548, 564, and 566, with their respectivechannels 538, 543, 552, 553, 562, and 565 are similar to blisters of thesame number of FIG. 15 of U.S. Patent Application No. 2010-0056383.Second-stage reaction zone 580 of FIG. 1 is similar to that of U.S.Patent Application No. 2010-0056383, but the second-stage wells 582 ofhigh density array 581 are arranged in a somewhat different pattern. Themore circular pattern of high density array 581 of FIG. 1 eliminatescorners and can result in more uniform filling of second-stage wells582. As shown, the high density array 581 is provided with 102second-stage wells 582. Pouch 510 is suitable for use in the FilmArrayinstrument. However, it is understood that the pouch embodiment isillustrative only.

Pouch 510 may be used in a manner similar to that described in U.S.Patent Application No. 2010-0056383. A 300 μl mixture comprising thesample to be tested (100 μl) and lysis buffer (200 μl) is injected intoinjection port (not shown) in fitment 590 near entry channel 515 a, andthe sample mixture is drawn into entry channel 515 a. Water is alsoinjected into a second injection port (not shown) of the fitment 590adjacent entry channel 515 l, and is distributed via a channel (notshown) provided in fitment 590, thereby hydrating up to eleven differentreagents, each of which were previously provided in dry form at entrychannels 515 b through 515 l via. These reagents illustratively mayinclude freeze-dried PCR reagents, DNA extraction reagents, washsolutions, immunoassay reagents, or other chemical entities.Illustratively, the reagents are for nucleic acid extraction,first-stage multiplex PCR, dilution of the multiplex reaction, andpreparation of second-stage PCR reagents, as well as control reactions.In the embodiment shown in FIG. 1, all that need be injected is thesample solution in one injection port and water in the other injectionport. After injection, the two injection ports may be sealed. For moreinformation on various configurations of pouch 510 and fitment 590, seeU.S. Patent Application No. 2010-0056383, already incorporated byreference.

After injection, the sample is moved from injection channel 515 a tolysis blister 522 via channel 514. Lysis blister 522 is provided withceramic beads and is configured for vortexing via impaction usingrotating blades or paddles provided within the FilmArray instrument.Once the cells have been adequately lysed, the sample is moved throughchannel 538, blister 544, and channel 543, to blister 546, where thesample is mixed with nucleic acid-binding magnetic beads. The mixture isallowed to incubate for an appropriate length of time, illustrativelyapproximately 10 seconds to 10 minutes. A retractable magnet locatedwithin the FilmArray instrument adjacent blister 546 captures themagnetic beads from the solution, forming a pellet against the interiorsurface of blister 546. The liquid is then moved out of blister 546 andback through blister 544 and into blister 522, which is now used as awaste receptacle. One or more wash buffers from one or more of injectionchannels 515 c to 515 e are provided via blister 544 and channel 543 toblister 546. Optionally, the magnet is retracted and the magnetic beadsare washed by moving the beads back and forth from blisters 544 and 546via channel 543. Once the magnetic beads are washed, the magnetic beadsare recaptured in blister 546 by activation of the magnet, and the washsolution is then moved to blister 522. This process may be repeated asnecessary to wash the lysis buffer and sample debris from the nucleicacid-binding magnetic beads.

After washing, elution buffer stored at injection channel 515 f is movedto blister 548, and the magnet is retracted. The solution is cycledbetween blisters 546 and 548 via channel 552, breaking up the pellet ofmagnetic beads in blister 546 and allowing the captured nucleic acids todissociate from the beads and come into solution. The magnet is onceagain activated, capturing the magnetic beads in blister 546, and theeluted nucleic acid solution is moved into blister 548.

First-stage PCR master mix from injection channel 515 g is mixed withthe nucleic acid sample in blister 548. Optionally, the mixture is mixedby forcing the mixture between 548 and 564 via channel 553. Afterseveral cycles of mixing, the solution is contained in blister 564,where a pellet of first-stage PCR primers is provided, at least one setof primers for each target organism, and first-stage multiplex PCR isperformed. If RNA targets are present, an RT step may be performed priorto or simultaneously with the first-stage multiplex PCR. First-stagemultiplex PCR temperature cycling in the FilmArray instrument isillustratively performed for 15-20 cycles, although other levels ofamplification may be desirable, depending on the requirements of thespecific application.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted, illustratively by forcing most of the sampleback into blister 548, leaving only a small amount in blister 564, andadding second-stage PCR master mix from injection channel 515 i.Alternatively, a dilution buffer from 515 i may be moved to blister 566then mixed with the amplified sample in blister 564 by moving the fluidsback and forth between blisters 564 and 566. If desired, dilution may berepeated several times, using dilution buffer from injection channels515 j and 515 k, and then adding second-stage PCR master mix frominjection channel 515 h to some or all of the diluted amplified sample.It is understood that the level of dilution may be adjusted by alteringthe number of dilution steps or by altering the percentage of the samplediscarded prior to mixing with the dilution buffer or second-stage PCRmaster mix comprising components for amplification, illustratively apolymerase, dNTPs, and a suitable buffer, although other components maybe suitable, particularly for non-PCR amplification methods. If desired,this mixture of the sample and second-stage PCR master mix may bepre-heated in blister 564 prior to movement to second-stage wells 582for second-stage amplification. Such preheating may obviate the need fora hot-start component (antibody, chemical, or otherwise) in thesecond-stage PCR mixture.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 102 second-stage wells 582 is pre-loadedwith a specific PCR primer pair. If desired, second-stage PCR master mixmay lack other reaction components, and these components may bepre-loaded in the second-stage wells 582 as well. Each primer pair maybe similar to or identical to a first-stage PCR primer pair or may benested within the first-stage primer pair. Movement of the sample fromblister 564 to the second-stage wells 582 completes the PCR reactionmixture. Once high density array 581 is filled, the individualsecond-stage reactions are sealed in their respective second-stageblisters by any number of means, as is known in the art. Illustrativeways of filling and sealing the high density array 581 withoutcross-contamination are discussed in U.S. Patent Application No.2010-0056383. Illustratively, the various reactions in wells 582 of highdensity array 581 are simultaneously thermal cycled, illustratively withone or more peltier devices, although other means for thermal cyclingare known in the art.

The illustrative second-stage PCR master mix contains the dsDNA bindingdye LCGreen® Plus to generate a signal indicative of amplification.However, it is understood that this dye is illustrative only, and thatother signals may be used, including other dsDNA binding dyes, andprobes that are labeled fluorescently, radioactively,chemiluminescently, enzymatically, or the like, as are known in the art.

The illustrative FilmArray instrument is programmed to make positive ornegative calls for each second-stage reaction based on a post-PCR melt.The melt curve must produce a melt peak (first derivative maximum ornegative first derivative maximum) within a pre-defined temperaturerange, for the call to be positive. It is understood that this method ofcalling each second-stage reaction is illustrative only, and that callscould be made using real-time amplification data or by other means, asare known in the art.

Example 1

The FilmArray Blood Culture Identification (BCID) system is designed toprovide rapid identification of a broad range of microorganism pathogensdirectly from blood culture. The illustrative BCID panel detects themost common bacteria and yeast isolated from positive aerobic bloodcultures (PABC), as well as select antibiotic resistance genes, with≥95% sensitivity. A commercial BCID panel is available from BioFireDiagnostics, Inc. This example uses a research version of the FilmArrayBCID panel to demonstrate methods of distinguishing between truepositives and environmental contamination.

Various gram-positive and gram-negative bacteria, as well as Candidayeast isolates were tested for assay reactivity. Mock PABC samples wereprepared by spiking microorganism into a mixture of human whole bloodand BD BACTEC Aerobic Plus/F blood culture medium. Microorganisms werespiked at concentrations consistent with that observed for blood culturebottles that had recently been indicated ‘positive’ for growth by the BDBACTEC 9050 system (103 to 108 CFU/mL)(Becton Dickinson, Franklin Lakes,N.J.). Exclusivity samples were prepared at microorganism concentrationsexpected for blood culture bottles that may have remained overnight (˜8hours after the initial positive signal) in a blood culture machine (108CFU/mL yeast and 1010 CFU/mL for bacteria). Samples were loaded into aFilmArray BCID pouch and processed in a FilmArray instrument. Nucleicacid extraction, purification, amplification, and results analysis areautomated using the FilmArray system, with a total processing time ofapproximately one hour.

PABC samples from children and adults from three different sites weretested in a FilmArray BCID pouch. FilmArray results were compared toconventional blood culture and susceptibility testing. One 250 μlaliquot from each PABC was mixed with 500 μl lysis buffer, and 300 μl ofthis mixture was loaded into a pouch per instructions and tested forgram positive and gram negative bacteria, fungi and antibioticresistance genes.

Within the FilmArray instrument, subsequent to sample prep, thefirst-stage multiplex PCR mixture was thermocycled in blister 564 from60° C. for 25 seconds to 96° C. for 4 seconds for 20 cycles. Afterfirst-stage PCR was complete, the mixture was diluted and wastransferred to each of the second-stage wells 582. The second-stage PCRreactions were subjected to 63° C. for 19 seconds to 94° C. for 0seconds for an additional 32 cycles. Melts in this illustrative examplewere performed after cycles 20, 26, and 32 for each second-stagereaction well 582 to generate melt curves, and each well was calledpositive if the melt curve showed a melt peak (negative first derivativeof the melt curve) in the pre-defined temperature range for eachsecond-stage assay. It is noted that other cycles may be used for meltanalysis, with 20, 26, and 32 cycles being illustrative only, and eachassay may have its own pre-defined temperature range that is related tothe Tm of the expected amplicon. The pre-defined temperature range worksto exclude amplified products that are non-specific such asprimer-dimers, which often will have a significantly different Tm. Fororganisms with variability in the target sequence, it may be desirableto have a wider pre-defined range, as sequence variability may result inslightly different Tms. For organisms with highly conserved targetsequences, it may be desirable to have a narrower pre-definedtemperature range, thus excluding most non-specific and cross-reactiveamplification.

FIGS. 2A-B show illustrative amplification and melting results for an A.baumannii test. FIG. 2A shows results for a contaminant that could leadto a false positive call, while FIG. 2B shows the results for a truepositive that was run after blood culture, as discussed above. It isnoted that each assay is run in triplicate in high density array 581 inthe illustrative BCID panel, and two of the three wells 582 must show apositive result for the system to call that organism positive. In FIG.2A, in two of the three replicates the amplification curve shows acrossing point (“Cp”) of 29.2. Thus, a call made before cycle 29,illustratively at cycles 20 or 26, would be negative, but a call madeafter cycle 29, illustratively at cycle 32, would be positive. This isconfirmed in the melts, where there is no melt peak after cycles 20(melt 1) and 26 (melt 2), but there is a clear melt peak after cycle 32(melt 3) for all three replicates, using a pre-defined temperature rangeof 78-83° C. Using either the amplification curve or the melt peaks,with the illustrative 20 or 26 second-stage amplification cycles, thisassay properly could have been called negative, but if PCR had gonethrough the illustrative 32 cycles, this assay could have resulted in afalse positive. In FIG. 2B, it is seen that the true positive amplifiedmuch earlier with a Cp between 7.9 and 8.0 for each well, and melt peaksat all three illustrative cycles would be called positive.

From FIGS. 2A-B, one may consider terminating the second-stageamplification at a cycle no later than cycle 26. Indeed, if A. baumanniiwere the only organism assayed, such would be a good strategy. However,a number of organisms in the BCID panel and in other panels amplify muchlater, illustratively because of slower growth in culture, lessefficient PCR, or because there are fewer copies of the target sequencein a positive blood culture. Fewer copies of the target sequence may bepresent because the organism is capable of triggering a positive bloodculture with fewer cells, or because there may be only one copy of thetarget sequence per cell, as compared to plasmid or RNA sequences thatmay be present in significantly higher copy numbers.

FIGS. 3A-B show illustrative amplification and melting results for a C.tropicalis assay. With this organism, true positives often do not showup until after cycle 26. With C. tropicalis, false positives would berare, but false negatives would be common if second-stage PCR wereterminated significantly earlier than cycle 32. If a single second-stagecycle were chosen for all assays, there would be either a risk of falsepositives for the assays that tend to have an earlier Cp (such as A.baumannii) or a risk of false negatives for assays that tend to have alater Cp (such as C. tropicalis), or both if a compromise cycle werechosen. Using different cycles for the calls for each of these organismsimproves the overall accuracy of the assay.

FIGS. 4A-B show the amplification and melting results for an S. aureusassay. With this organism in the FilmArray BCID panel, true positivessometimes show up as early as cycle 20. FIG. 4B shows that all threereplicates were called negative after 20 cycles by Cp, but one replicatewas called positive by melt. However, all three replicates were calledpositive after 26 cycles by Cp and melt. While the true negative shownin FIG. 4A did not show any amplification, even after 32 cycles, it isknown that S. aureus is a moderate contamination risk. Accordingly,while choosing cycle 32 may be acceptable based on the data shown inFIGS. 4A-B, cycle 26 is also a viable choice, with less risk of falsepositives from environmental contamination.

Each organism in the illustrative BCID panel was analyzed to determinewhether melt cycle 1 (second-stage PCR cycle 20), melt cycle 2(second-stage PCR cycle 26), or melt cycle 3 (second-stage PCR cycle 32)would be most appropriate to use to minimize both false positives andfalse negatives. The organisms were assigned as follows in Table 1:

TABLE 1 Melt Cycle 1 Melt Cycle 2 Melt Cycle 3 (Cycle 20) (Cycle 26)(Cycle 32) A. baumannii Enterococcus K. pneumoniae C. albicans E. coliL. monocytogenes K. oxytoca C. glabrata E. cloacae Staphylococcus S.marcescens C. krusei Enterobacteriaceae S. aureus P. aeruginosa C.parapsilosis Proteus Streptococcus N. meningitidis C. tropicalis S.agalactiae mecA S. pneumoniae KPC S. pyogenes vanA/B H. influenzae

In the illustrative embodiment, the FilmArray instrument was programmedto collect the melt result for each organism only in the melt cyclelisted above. While only the melt cycle identified in Table 1 was usedfor each organism, it is understood that obtaining amplification or meltpeak information over multiple cycles for a single well may be useful insome circumstances.

In general, melt cycle 1 targets are present at the highest titers inpositive aerobic blood cultures, but also present as backgroundorganisms and are the highest risk for unexpected positives. Melt cycle2 targets present at high titers in positive aerobic blood cultures, buthave a low presence as background organisms and are a medium risk forunexpected positives. Melt cycle 3 targets present at low titers inpositive aerobic blood cultures, but also have low to no presence asbackground organisms and have a low risk for unexpected positives.

When the three melts discussed above were used, it was found that theillustrative version of the FilmArray BCID panel exhibited 100%reactivity (111/111) with the panel of inclusivity microorganisms(including those harboring antimicrobial resistance genes). For example,the illustrative FilmArray BCID panel detected 17/17 Staphylococcusisolates, 19/19 Enterococcus isolates, and 30/30 Enterobacteriaceaeisolates. Similarly, the illustrative FilmArray BCID system did notdetect 62/62 (100%) microorganisms for which the assays were notexpected to react. The average specificity per interpretation [TrueNegative/(True Negative+False Positive)] in the BCID system was 100%(155/155; 95% CI 98.1-100.0%). These results demonstrate that each wellmay be called correctly using only a single melt cycle for thatreaction, which may be different than the single melt cycle used for thereaction in another well in the same assay.

While three melt cycles were used in this example, it is understood thatany number of melt cycles may be used and that any cycle may be chosenas a melt cycle. Separation between false positives and false negativesmay be achieved with only two melt cycles in some assays, whereas fouror more melt cycles may be needed in other assays. Further, while theexample used samples from culture, it is understood that multiple meltcycles may be appropriate for assays using uncultured materials.Further, while melting is used in this example, amplification curveswith cut-offs or Cps at the various cycles may be used to determinewhether the sample is positive for the target.

Additionally, it is understood that the information obtained for oneorganism can be used to assist with positive or negative calls for otherorganisms, particularly if there is some cross-reactivity between theorganisms, or if there is some other relationship such as a bacteriumand an antibiotic resistance gene associated with that bacterium. In theabove example, Enterococcus (“Entero”) and Staphylococcus (“Staph”) areboth detected in melt cycle 2. However, in many known assays for Entero,due to similarities in target sequence, there is cross-reactivity withStaph, thereby potentially causing a late Cp in a true negative Enterosample that is positive for Staph. To reduce the effect of potentialcross-reactivity for the Entero assay in such a situation wherecross-reactivity is an issue, a positive or negative call for Staph maybe made, illustratively using melt cycle 2 (cycle 26). If Staph ispositive, thereby affecting the Entero sample, Entero could be calledbased on an earlier result, illustratively melt cycle 1 (cycle 20). IfStaph is negative, then the Entero assay would be unaffected and thecall may be made illustratively at melt cycle 2, or whichever cycle waschosen as optimized for that assay without cross-reactivity. It isnoted, however, that in blood culture, a positive bottle ring is basedon the combined organism growth of all organisms that are present, andone or more organisms may be present at amounts lower than either wouldbe from a single infection. The cycle at which the cross-reactive assayis called may need to be adjusted accordingly. By adjusting the cycleused for the call of the cross-reactive assay based on a positive ornegative call from the other assay, cross-reactivity issues from doubleinfection samples can be called accurately, illustratively without theneed to redesign the primers to avoid cross-amplification.

It is understood that, while the above example identifies organisms, itis understood that the same methods and devices may be used to identifydifferent target sequences in one or several organisms by amplifyingdifferent loci of that organism.

Example 2

In Example 1, melts at different cycle numbers were used to distinguishbetween environmental contamination and clinical infection, wherein eachtest in the panel was assigned a cycle number, and positives andnegatives were called based on the result at the assigned cycle number.Using different cycle numbers for calls can also be used to distinguishbetween potential “false positives” where nucleic acid is present atsubstantial quantities but not clinically relevant and clinicallyrelevant true positives that do not have a crossing point until a latercycle. One such example is with latent viral infection throughchromosomal integration, wherein the chromosomally integrated viral DNAmay or may not be responsible for the clinical symptoms.

For example, an individual may have inherited the HHV6 virus from aparent who had been infected with the virus and the virus was latentlychromosomally integrated (termed chromosomally-integrated HHV6,“ciHHV6”). This individual would have some or all of the HHV6 virusintegrated in essentially every nucleated cell, and a PCR test for HHV6would always come up positive, even if the individual has a latentinfection with no active clinical symptoms from that virus. For such apatient with no active symptoms from that virus, the integrated viralchromosome would not be clinically relevant, and any symptoms would befrom some other source.

For HHV6 patients who have an active case of meningitis and do not haveciHHV6 virus (hereinafter “clinically-relevant infection”), it isexpected that a FilmArray second-stage crossing point from a spinalfluid sample would be around cycle 25-30, while a meningitis patienthaving a latent ciHHV6 virus would have a FilmArray second-stagecrossing point around cycle 6-10. In such a situation, the first meltcycle could be illustratively around cycle 10, and a later melt cyclecould be done illustratively around cycle 30. However, it is understoodthat these cycles are illustrative only and other cycles may beappropriate. If the first melt cycle were positive, the test may reporta “negative”, or it may report a “chromosomal integration” or some otherresult indicative of the early cycle positive result. Of course, if thefirst melt cycle were positive, the later melt cycle would also bepositive. However, if the first melt cycle were negative and the secondmelt cycle were positive, this would be an indication of currentinfection, and a “positive” result would be reported. Thus, in somecases, an early cycle “positive” can be used to identify anon-clinically relevant positive result.

Example 3

In Examples 1 and 2, different cycle numbers were used to distinguishbetween environmental contamination, potentially non-clinically relevantinfection, and clinically-relevant infection. In this example,additional cycles are used to enable detection of low level truepositives. In this method, the detection and identification method is amodified two-step process. The first step is a set amplificationprotocol, optionally with additional melt cycles as used in Examples 1and 2, and the second step employs a higher signal-to-noise detectionduring at least one subsequent melt. An illustrative protocol is shownin FIG. 5.

As shown in FIG. 5, a set number of amplification cycles, illustratively26 cycles, are run. Any wells that return a positive result at cycle 26optionally need not be analyzed further. The positive result may be madeby amplification curve, or may be made or confirmed by a melt curveanalysis as discussed above, for those samples that show amplification,illustratively by exceeding a threshold fluorescence level as indicatedby the High-Con calls in FIG. 6, or other methods as are known in theart. Thereafter, optionally a melt is run during each of a plurality ofadditional cycles. After each cycle, if a melt peak is detected, theshape of the amplification curve optionally may be analyzed for furtherconfidence of the positive calls. Illustrative methods of makingpositive calls from the shape of amplification curves may be found inU.S. Pat. Nos. 6,387,621; 6,730,501; and 7,373,253, and U.S. PatentApplication No. 2011-0238323, all of which are herein incorporated byreference. Such methods may aid in distinguishing true amplificationfrom signal drift, which is particularly useful with low levelpositives. After these additional cycles, illustratively after cycle 30,the light source, illustratively an LED although other excitationdevices may be used, in the instrument is adjusted to increase thepower. After adjusting the LED power, the instrument collectsfluorescence data during a melt. This power adjustment is made toincrease the signal-to-noise ratio for detecting low load samples. Areason for not going to full power for the initial melt is that this mayhave the effect of railing the signal from one or more sample wells thatwere called positive after cycle 26, as these samples already had asignificant load. However, data collection from these wells optionallywould be terminated after the positive call at cycle 26, so the railingwould not have any effect on reported results. Finally, a melt curveanalysis (amplification detection as described above and/or and Tmidentification) is performed on all reactions with a cycle 26 or cycle30 end-point fluorescence less than the established threshold, todetermine whether any of these sample wells contain a true positiveresult.

It is understood that the use of cycles 26 and 30 is illustrative only,and that other cycles may be used, as may be desired for the specificapplication. Furthermore, the additional cycles 27-30 may be omitted,and the light source may be adjusted after the initial amplification.

Example 4

Optionally, instead of or in addition to multiple melting cycles, thelight source in the instrument, illustratively an LED, although otherexcitation devices may be used, may be adjusted for different assays.The data in Table 2 show that if the LED power is reduced, therebyreducing the fluorescence signal, the detection of background bacterialorganisms can be reduced. In one illustrative example, reducing the LEDpower from 70% (approximate current FA setting) to 50% reducedunexpected false positive detection by the FA BCID Enterobacteriaceaetest from 90% to 20% of tests after 32 cycles.

TABLE 2 Historical Background FA 10% 30% 50% 70% 90% detection BCID LEDLED LED LED LED (65% LED Assay Power Power Power Power Power Power)Abaumannii 0/10 1/10 0/10 0/10  0/10 2.17%  (0%) (10%)  (0%)  (0%)  (0%)Ecloacae 0/10 0/10 0/10 0/10  0/10 8.70%  (0%)  (0%)  (0%)  (0%)  (0%)Ecoli 0/10 0/10 0/10 4/10  5/10 23.40%  (0%)  (0%)  (0%) (40%) (50%)Enterobacteriaceae 0/10 5/10 2/10 9/10 10/10 78.72%  (0%) (50%) (20%)(90%) (10%) mecA 0/10 0/10 2/10 0/10  0/10 22.58%  (0%)  (0%) (20%) (0%)  (0%)

While an illustrative setting is 70% LED power, a single setting may ormay not be appropriate for all assays, and it is understood that theideal LED power may be different for various assays within an array orpanel. For example, an assay that is more susceptible to false positivesfrom environmental contamination may be better off with a lower powersetting to reduce sensitivity, while an assay that for which low-levelpositives are important may benefit from higher LED power. Thus, afterthe individual positive or negative calls are made, the LED power may bereduced, illustratively by 5%, 10%, 15% or more or any other level, anda melt curve generated. If the melt curve is negative, that assay may beflagged as a potential false positive, or it may be reported as anegative. Alternatively or additionally, the LED power may be increased,illustratively by 5%, 10%, 15% or more or any other level, and assaysthat were previously called negative may be interrogated, withsubsequent melt curves potentially indicated a positive result for alow-level assay.

While LEDs and LED power is discussed herein, it is understood thatother illumination sources may be used, including incandescent,fluorescent, and other lamps, and adjustment of the power andconcomitant lighting output of such lamps is also within the scope ofthis invention.

Example 5

As an extension of the previous examples, melt curves may be acquiredduring additional cycles, for example at every cycle or nearly everycycle of PCR, illustratively through continuously acquired temperatureand fluorescence data during amplification. For example, an illustrativetwo-step PCR protocol may be divided two segments: adenaturation/annealing segment where the temperature is constantlychanging, and an extension segment where the temperature is heldconstant. During the denaturation/annealing segment, the temperature ofthe PCR reaction is increased, illustratively at a constant rate, from abaseline value to a maximum temperature value, followed by a rapiddecrease in the temperature back to the baseline value. As thetemperature is increased, the dsDNA is separated into two ssDNAfragments. As the temperature is decreased, the PCR primers anneal tothe two ssDNA fragments. During the extension segment, the temperatureis held constant at the baseline value, allowing the primed ssDNAfragments to extend to form two dsDNA fragments. FIG. 7 is a graphicaldepiction of this illustrative temperature cycling protocol. However, itis understood that other protocols may be used, wherein the temperatureis held constant at any or all of the melting temperature, the annealingtemperature, and the elongation temperature, or without any holds. Also,it is understood that the baseline annealing temperature may be the sameas or different from the extension temperature.

With continuous data acquisition, an instrument may collect temperatureand fluorescence data during both segments of the PCR protocol,continuously for all cycles, as shown in FIG. 8. Fluorescence datacollected as part of the denaturation segments can be thought of as aseries of melt curves. In between each melt curve, the fluorescence datashows amplification as the PCR product is extended and non-specificdsDNA-binding dyes may be used to detect the amplification product (seeFIG. 9). At the start of PCR cycling, the amount of dsDNA is low and,therefore, the signal generated during the denaturation segment, is alsolow. As cycling continues, the amount of PCR product begins to increase.Similarly, the signal generated by the denaturation segment alsoincreases. See FIG. 10 for a graphical depiction of the change to meltcurves as PCR cycling progresses.

One method for quantifying a target nucleic acid is by determining Cpand comparing the Cp to a standard or to a control. As an alternative todetermining Cp by absolute or normalized amplification data, the seriesof melt curves discussed above may be used. FIG. 11 shows anillustrative set of negative derivative melt curves, wherein theflattest curves represent the earliest cycles and the area under thecurve increases through a number of cycles. It is expected that suchderivative melt curves acquired at a plurality of cycles duringamplification can be used to determine Cp. In this illustrative example,the height of the transition for each melt curve or the area under thenegative first derivative of the melt curve can be determined for eachcycle. The Cp may then be assigned to the cycle at which this valueexceeds a pre-determined threshold. It is understood that every cyclemay be used, or fewer than all cycles may be used for an approximate Cp.

Additional methods for determining Cp may be applied. For example, amelt detector may be used (see U.S. Pat. Nos. 6,387,621; 6,730,501; and7,373,253, herein incorporated by reference). The detector wouldinterrogate curve shape and background noise to determine if PCR productis present in the sample. The use of a melt detector could be used toincrease the sensitivity of the system (See Poritz, et al., PLos One6(10): e26047). Optionally, additional filters could be applied to themelt curve analysis to window the melt transition to increase thespecificity of the system, by analyzing only those melt curves having amelting transition, illustratively displayed as a melt peak, within aset temperature range. It is expected that such methods would result ina more accurate Cp.

Example 6

In another illustrative example, methods of continuous monitoring oftemperature and fluorescence are used for relative quantification,illustratively using a dsDNA-binding dye in a single reaction with acontrol nucleic acid. In this example, a multiplexed PCR reaction isprovided, containing a control nucleic acid at a known initialconcentration and a target nucleic acid at an unknown concentration.Illustratively, primers for amplification of the control nucleic acidare present at the same initial concentration as primers foramplification of the target nucleic acid. In addition, it is desirableif the control nucleic acid is selected such that its meltingtemperature is sufficiently well separated from the melting temperatureof the target nucleic acid, so that melting of each of these nucleicacids is discernable from melting of the other. It is understood thatmultiple target nucleic acids of unknown concentration may bemultiplexed in the reaction, noting that it is desirable that the meltcurve for each nucleic acid is distinguishable from the others and fromthe control nucleic acid.

In an illustrative PCR application, the amplification of the controlnucleic acid and the target nucleic acid produce an amplification curvesimilar to that shown in FIG. 12. In such a curve generated using adsDNA binding dye, signal from the control and the target combine togenerate a single amplification curve as shown, and information aboutthe amplification of the individual nucleic acids is not discernable.

With continuous data acquisition, a series of melt curves are generatedduring PCR cycling. Provided that the melting temperatures of thecontrol nucleic acid and the target nucleic acid are sufficientlyseparated, the melting profile of each of the two reactions can bedistinguished, as shown in FIG. 13. Illustratively, an adjustedamplification curve for the target nucleic acid and optionally for thecontrol nucleic acid can be generated from this series of melt curves.Illustratively, to generate a corrected amplification curve for thecontrol nucleic acid, at each cycle the integral of the negative firstderivative of the melt curve over a pre-defined melt window can becomputed and plotted as a function of the cycle number, with the Cpdetermined as the cycle at which each value exceeds a predeterminedvalue. Similarly, a corrected amplification curve for the target nucleicacid may be generated by integrating the negative first derivative ofthe melt curve over the pre-defined melt window for the target. FIG. 13shows illustrative negative derivative melt curves for 5, 10, 15, 20,and 25 cycles, with illustrative pre-defined melt windows indicated forthe two nucleic acids. Such corrected amplification curves areillustrated in FIG. 14. Other methods for converting the melt curve to avalue are known in the art, illustratively using peak height of thenegative first derivative. It is understood that the predetermined valueshould be selected according to method used.

The concentration of the target nucleic acid relative to the controlnucleic acid may be computed using the formula:

Relative Concentration=ET*Cp,t/EC*Cp,c  [Equation 1]

where

-   -   ET and EC are the target and control efficiencies, and    -   Cp,t and Cp,c are the target and control crossing points.

The efficiency of the two reactions may be determined empirically andthe Cp values for the two reactions may be computed using standardcalculations on the amplification curves computed from the series ofmelt curves, as is known in the art.

Example 7

Certain embodiments of the present invention may also involve or includea PCR system configured to make positive or negative calls fromamplification curves or melt curves or a combination thereof.Illustrative examples are described in U.S. Patent Application No.2010-0056383, already incorporated by reference, for use with pouch 510or similar embodiments. However, it is understood that the embodimentsdescribed in U.S. Patent Application No. 2010-0056383 are illustrativeonly and other systems may be used according to this disclosure. Forexample, referring to FIG. 15, a block diagram of an illustrative system700 that includes control element 702, a thermocycling element 708, andan optical element 710 according to exemplary aspects of the disclosureis shown.

In at least one embodiment, the system may include at least one PCRreaction mixture housed in sample vessel 714. In certain embodiments,the sample vessel 714 may include a PCR reaction mixture configured topermit and/or effect amplification of a template nucleic acid. Certainillustrative embodiments may also include at least one sample block orchamber 716 configured to receive the at least one sample vessel 714.The sample vessel 714 may include any plurality of sample vessels inindividual, strip, plate, or other format, and, illustratively, may beprovided as or received by a sample block or chamber 716.

One or more embodiments may also include at least one sample temperaturecontrolling device 718 and/or 720 configured to manipulate and/orregulate the temperature of the sample(s). Such a sample temperaturecontrolling device may be configured to raise, lower, and/or maintainthe temperature of the sample(s). In one example, sample controllingdevice 718 is a heating system and sample controlling device 720 is acooling system. Illustrative sample temperature controlling devicesinclude (but are not limited to) heating and/or cooling blocks,elements, exchangers, coils, radiators, refrigerators, filaments,Peltier devices, forced air blowers, handlers, vents, distributors,compressors, condensers, water baths, ice baths, flames and/or othercombustion or combustible forms of heat, hot packs, cold packs, dry ice,dry ice baths, liquid nitrogen, microwave- and/or other wave-emittingdevices, means for cooling, means for heating, means for otherwisemanipulating the temperature of a sample, and/or any other suitabledevice configured to raise, lower, and/or maintain the temperature ofthe sample(s).

The illustrative PCR system 700 also includes an optical system 710configured to detect an amount of fluorescence emitted by the sample 714(or a portion or reagent thereof). Such an optical system 710 mayinclude one or more fluorescent channels, as are known in the art, andmay simultaneously or individually detect fluorescence from a pluralityof samples.

At least one embodiment of the PCR system may further include a CPU 706programmed or configured to operate, control, execute, or otherwiseadvance the heating system 718 and cooling system 720 to thermal cyclethe PCR reaction mixture, illustratively while optical system 710collects fluorescent signal. CPU 706 may then generate an amplificationcurve, a melt curve, or any combination, which may or may not beprinted, displayed on a screen, or otherwise outputted. Optionally, apositive, negative, or other call may be outputted based on theamplification and/or melt curve. Optionally only the calls areoutputted, illustratively one call for each target tested.

Additional examples of illustrative features, components, elements, andor members of illustrative PCR systems and/or thermal cyclers(thermocyclers) are known in the art and/or described above or in U.S.patent application Ser. No. 13/834,056, the entirety of which is hereinincorporated by reference.

Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

1. (canceled)
 2. A method for analyzing a target nucleic acid in asample comprising (a) providing a sample container comprising a firstsample well and a second sample well, the second sample well comprisingtherein primers for amplifying the target nucleic acid, (b) subjectingthe sample to a first amplification in the first sample well, and movinga portion of the sample from the first sample well to the second samplewell, (c) subjecting the second sample well to amplification conditionsthrough a plurality of amplification cycles to amplify the targetnucleic acid in the second sample well, (d) generating a melt curve ofthe amplified target nucleic acid in the second sample well, (e)repeating steps (c) and (d), and (f) determining a Cp by identifying theamplification cycle in which a value for the melt curve exceeds apredetermined value, wherein the sample in the second sample wellcontains two or more different target nucleic acids and the secondsample well contains primers for amplifying the two or more differenttarget nucleic acids, and the method further comprises: simultaneouslyamplifying the two or more target nucleic acids in the second samplewell through a plurality of amplification cycles, generating a pluralityof melt curves of the amplified two or more target nucleic acids,determining a value for each of the plurality of melt curves from eachof the amplified two or more target nucleic acids, and determining amelt Cp for each of the two or more target nucleic acids by identifyinga melt event in which the value for the melt curve for each of the twoor more target nucleic acids exceeds a predetermined value.
 3. Themethod of claim 2, wherein the first amplification of step (b) is amultiplex amplification.
 4. The method of claim 2, wherein the value forthe melt curve is determined by peak height or peak area of a negativederivative of the melt curve.
 5. The method of claim 2, furthercomprising generating the melt curve at every amplification cycle ornearly every amplification cycle.
 6. The method of claim 2, wherein thetwo or more target nucleic acids amplified in the second well areselected such that a melting temperature for the two or more targetnucleic acids are sufficiently well separated from one another, so thatmelting of each one of the two or more target nucleic acids isdiscernable from melting of the others.
 7. The method of claim 2,wherein different amplification cycle numbers predetermined for each ofthe two or more target nucleic acids are used to distinguish betweentrue positive and false positive detections.
 8. The method of claim 2,wherein different amplification cycle numbers predetermined for each ofthe two or more target nucleic acids are used to distinguish betweenclinically relevant infection, environmental contamination, orchromosomal integration.
 9. A method for analyzing a target nucleic acidin a sample comprising (a) providing a sample container comprising afirst sample well and a second sample well, the second sample wellcomprising therein primers for amplifying the target nucleic acid, acontrol nucleic acid, and primers for amplifying the control nucleicacid, (b) subjecting the sample to a first amplification in the firstsample well, and moving a portion of the sample from the first samplewell to the second sample well, (c) subjecting the sample well toamplification conditions through at least one amplification cycle toamplify the target nucleic acid and the control nucleic acid in thesample well, wherein the amplification conditions include a dsDNAbinding dye, (d) generating a melt curve of the amplified target nucleicacid and amplified control nucleic acid, (e) determining a value for theamplified target nucleic acid, and (f) repeating steps (c), (d), and(e).
 10. The method of claim 9, wherein the second sample well furthercomprises the dsDNA binding dye.
 11. The method of claim 9, wherein step(e) also includes determining a value for the amplified control nucleicacid.
 12. The method of claim 11, further comprising the step ofgenerating a corrected amplification curve for the target nucleic acidusing the values determined in step (e).
 13. The method of claim 12,wherein the melt curves are plotted as negative derivative melt curves,and the values are determined by peak height or peak area of a selectedtemperature window for each of the target and control nucleic acids. 14.The method of claim 13, further comprising the step of determining arelative starting concentration of the target nucleic acid relative to astarting concentration of the control nucleic acid.
 15. The method ofclaim 14, wherein the relative starting concentration is determinedusing the formulaET*Cp,t/EC*Cp,c where ET and EC are target and control efficiencies, andCp,t and Cp,c are target and control crossing points, determined by acycle at which the value for the amplified template nucleic acid exceedsa predetermined value and at which the amplified control nucleic acidexceeds the predetermined value.
 16. The method of claim 9, furthercomprising the step of generating a corrected amplification curve forthe control nucleic acid.
 17. The method of claim 9, further comprisinggenerating the melt curve at every amplification cycle or nearly everyamplification cycle.
 18. The method of claim 9, wherein sample containercomprises a plurality of second sample wells for amplification of aplurality of target nucleic acids and wherein one or more of theplurality of second sample wells includes the control nucleic acid. 19.The method of claim 18, wherein some of the plurality of sample wellsinclude different control nucleic acids configured to be amplified withone or more of the plurality of target nucleic acids, and wherein thedifferent control nucleic acids are selected such that their meltingtemperatures are separate and distinguishable from the meltingtemperatures of the one or more of the plurality of target nucleicacids.
 20. The method of claim 10, wherein different amplification cyclenumbers predetermined for the target nucleic acid are used todistinguish between true positive and false positive detections.
 21. Themethod of claim 10, wherein different amplification cycle numberspredetermined for the target nucleic acid are used to distinguishbetween clinically relevant infection, environmental contamination, orchromosomal integration.